Nasal microbiome and the effect of nasal decolonization with a novel povidone-iodine antiseptic solution: a prospective and randomized clinical trial

The aim of this study was to assess the profile of nasal microbiome and evaluate the effect of a specific nasal decolonization solution on the microbiome. We conducted a randomized, placebo-controlled, and parallel-group clinical study of 50 volunteers aged 18 years and older. The subjects were randomly assigned to receive a nasal antiseptic solution, containing povidone-iodine as the main ingredient, (n = 25) or a control solution (n = 25). Nasal swabs were obtained before application (baseline) and at 3 timepoints after application (5 min, 2 h, 24 h). Nasal swabs were subjected to next generation sequencing analysis and cultured in agar plates. At baseline, there were substantial associations between anaerobic species, Corynebacterium spp., Staphylococcus spp., and Dolosigranulum spp. Then, a high bioburden reduction was observed after the application of povidone-iodine (log10 3.68 ± 0.69 at 5 min; log10 3.57 ± 0.94 at 2 h; log10 1.17 ± 1.40 at 24 h), compared to the control. The top species affected by the treatment were Cutibacterium acnes, Staphylococcus, and Corynebacterium species. None of the subjects experienced any adverse effects, nor increases in mucociliary clearance time. Antiseptic solutions applied to the anterior nares can transiently and markedly reduce the bioburden of the nose. The registration number for this clinical trial is NCT05617729.


Bioburden reduction
The baseline nose bioburden varied considerably among individuals (3517.5 [IQR, 398.5-13,176]CFU/ml).At 5 min after application of the nasal antiseptic solution, we observed a higher and almost a complete elimination of bacteria from the anterior nares, compared to PBS (log 10 reduction after PVP-I 3.68 ± 0.69 vs. log 10 reduction after PBS 0.94 ± 1.12; t statistic, p < 0.01; Fig. 2a).At 2 h after the application of PVP-I, bacterial reduction of log 10 3.57 ± 0.94 (t statistic, p < 0.01) was still observed.We assessed bioburden at baseline, 5 min, 2 h, and 24 h post-application, meanwhile microbial diversity and mucociliary clearance was assessed at baseline and 24 h post-application.Bacterial associations were assessed at baseline and non-desired events were examined at every follow-up visit.Abbreviations: PBS; phosphate-buffered saline, PVP-I; povidone-iodine.
Moreover, by 24 h, the reduction in bacterial load was still detected in individuals receiving PVP-I (log 10 reduction after PVP-I 1.17 ± 1.40 vs. log 10 reduction after PBS 0.33 ± 1.44; t statistic, p = 0.14).The bacterial load analysis by 16S qPCR supported the findings from culture, suggesting a major difference between baseline and 24 h post-application, especially after the use of the PVP-I formulation.The mean reduction after 24 h was 0.67 in the PVP-I group (Tukey test, mean difference from baseline − 0.67 [95% CI − 1.21, − 0.14], adjusted p = 0.01) and 0.25 after applying PBS (Tukey test, mean difference from baseline − 0.25 [95% CI − 0.77, 0.26], adjusted p = 0.56).

Bacterial associations at baseline
A total of 18 paired species associations were marked as being found in co-occurrence at a rate different from expected by random chance (Spearman ranked correlations, p < 0.05) whereas an additional 12 species were approaching significance (Spearman ranked correlations, p < 0.1; Fig. 4 and Table 1).From the binary perspective, the greatest positive associations were observed among a set of primarily anaerobic species, including Finegoldia magna, Anaerococcus, and Corynebacterium species.Moreover, C. accolens and Dolosigranulum pigrum were negatively associated with S. aureus (Spearman ranked correlations, r = − 0.151, p = 0.031) and Staphylococcus epidermidis (Spearman ranked correlations, r = − 0.387, p = 0.027) presence, respectively.Furthermore, when considering relative abundance, a similar positive trend was observed among anaerobic bacteria (Fig. 5; Spearman ranked correlations, p < 0.05).Overall, Staphylococcus and Corynebacterium species' abundance showed a positive correlation; however, S. aureus' abundance was negatively correlated with both C. accolens and D. pigrum.S. epidermidis' abundance showed the most significant positive correlation with anaerobic species.

Fungi diversity
Fungi was detected by NGS analysis in half of the samples at baseline.This rate was not modified after the application of PVP-I.The principal fungi detected in our study were Malassezia species, with a relative abundance of nearly 80% throughout all samples.The application of PVP-I did not disturb the number of OTUs (Tukey test, mean difference from baseline − 7.0 [95% CI − 21.44, 7.44], adjusted p = 0.51) nor Hill1 diversity (Tukey test, mean difference from baseline − 0.19 [95% CI − 3.37, 3], adjusted p > 0.99).Moreover, the fungi composition did not vary at the 24-h timepoint, according to the weighted and unweighted UniFrac PCA.detected to be differentially abundant between timepoints by ANCOMBC procedure.A cell colored in black can be considered "true" zero, whereas there were no detections of a particular bacteria for that given cell.This plot was performed using R (version 3.0.1,http:// www.r-proje ct.org), by the RTL Genomics facility (Lubbock, Texas).Abbreviations: PVP-I; povidone-iodine.www.nature.com/scientificreports/

Mucociliary function and adverse effect assessment
None of the subjects in this study experienced adverse events at any timepoint during the study.Moreover, the mucociliary clearance (MCC) assessment demonstrated that application of PVP-I did not slow the mucociliary function as both groups detected sweetness in a similar timeframe in all time points (Fig. 2b).After 24 h of the nasal PVP-I application, we observed a median of almost a minute and a half to sweetness detection (1.

Discussion
It is well known that nasal and extra-nasal surfaces harbor microorganisms that can cause SSIs 5,6,13 .Thus, nasal decolonization has been shown to be an effective strategy to reduce the bioburden in the nares and impact the rate of SSIs 5,14,15 .Surprisingly, there is little data on the safety and effect of nasal decolonization on the nose bioburden 5,16,17 and microbial composition 18,19 .
The current study provides a great insight into the nasal microbiome and the effect that a novel povidoneiodine (PVP-I) antiseptic solution had on the bioburden of the nares.An independent formulation was used to avoid confounding related to excipients in commercially available products.Moreover, testing the active ingredient in an independent formulation may enhance the embracement of our results by healthcare systems and the research community.
Using next generation sequencing (NGS), we were able to map out the microbiome composition of the nares.To note, our study targeted the hypervariable regions V1-V2.This is important, since previous studies have demonstrated that the targeted sequence strongly influenced the microbial communities captured by NGS [20][21][22] , and should be selected based on the sample being studied (e.g., skin, nose, synovial fluid, stool, among others) 20,21,23,24 .The V1-V3 region has shown the highest ability to discriminate Staphylococcus species 20,22 and a fairly similar performance has been observed with the V1-V2 region 21 , with a shorter sequence length.Moreover, it is plausible that these primers gave us a lower discrimination power for Pseudomonas and Neisseria species 22 , which should be taken in consideration in further comparisons.
The anterior nares of individuals in our study were mainly comprised of Staphylococcus, Cutibacterium, and Corynebacterium species.The nose mucosa, a transition area between dry and moist areas 6 , has shown a similar composition to sebaceous and moist skin areas, with a remarkable presence of species belonging to these 3 geni 7 .Nearly 25% of individuals in our study harbored S. aureus in the anterior nares 11,12 .
We were also interested in studying the inter-organism interaction.Using bioinformatics, we detected that presence of Corynebacterium spp., was negatively associated with S. aureus colonization, perhaps indicating an inter-species competition 25,26 .In addition, Dolosigranulum spp.and the Gammaproteobacteria taxon were identified as informative predictors for S. aureus non-carriage 12,25,27 .Dolosigranulum spp can also affect coagulasenegative Staphylococcus species, as demonstrated in our study.Notably, we detected S. aureus abundance was negatively correlated with both C. accolens and D. pigrum abundance.Still, the specific mechanisms facilitating or interfering with the microbial interactions in a nutrient-limited mucosa, remain an area of interest to scientists.www.nature.com/scientificreports/ The international community has also focused research on microbial interactions within the nose microbiome 12,25,26,28,29 .A recent review thoroughly addressed the nutritional interactions between species of the nose microbiome, pointing out that anaerobic microniches are favored by crypts and the nearby presence of aerobe species, which ultimately deplete oxygen 28,30 .In this regard, our study detected the strongest binary positive associations between Corynebacterium spp and a set of anaerobic species at baseline.Moreover, when considering relative abundance, Corynebacterium and coagulase-negative Staphylococcus species, mainly S. hominis, were also positively correlated.Both facts, later supported the positive correlation observed between the abundance of S. epidermidis and anaerobic species in our study.
The nose microbiome composition barely relies on host genetics 25 , bringing up the possibility of manipulation by environmental factors.In our study, species belonging to the most abundant genus at baseline were found to vary post-application, even with PBS.The mechanical force of the swab, plus the diluent effect of PBS could explain this latter fact.To note, a detachment of microorganisms was observed in the PBS group (mean log 10 reduction of 0.94 at 5 min).After a single application of PVP-I, our findings suggested the reduction of pathogenic (S. aureus) and easily available bacteria (Corynebacterium spp.), and enhancement of more indolent species (anaerobic and coagulase-negative Staphylococcus).
Our study is the first to elucidate the effect of a PVP-I based formulation on the nose microbiome.Previous studies have examined the effect of different antimicrobials on nose microbiome 18,19 .After a dual application for 5 days of a mupirocin ointment, Roghmann et al. also found a reduction in the presence and abundance of S. aureus.Similar to our results, a regrowth of coagulase-negative Staphylococcus species was observed.Nevertheless, the increasing resistance rates associated with this compound pose a major drawback to the extensive use of mupirocin 17,31 .In our study we noted that a single application of PVP-I solution resulted in www.nature.com/scientificreports/ a significant bioburden reduction even after 5 min and part of this effect lasted as long as 24 h.The effect of PVP-I appeared to be organism specific also.For example, the relative frequency of C. accolens decreased while the relative and absolute frequency of S. epidermidis was less affected.C. accolens depicts a controversial role, since previous authors have pointed out a protective and cooperative role in S. aureus carriage 12,26,32,33 .It is also interesting to note that regrowth was mainly attributed to S. hominis and C. acnes and the application of PVP-I did not disturb the fungal composition in the anterior nares.
Our study also evaluated the effect of PVP-I on mucociliary function and noted that MCC was not at all disrupted.This is important as nasal mucosa plays a critical defense role against pathogens 34,35 .Paralysis of ciliary function of nasal mucosa by an antiseptic agent would be problematic.
Our study has some limitations.First, the culture conditions prioritized aerobic bacteria over strictly anaerobic bacterial and fungal species.Most of these microorganisms are fastidious and difficult to grow even under appropriate culture conditions 36 .This may have required more swabs per timepoint, thus repeated mechanical rubbing, and more complex sampling techniques.We did, however, use targeted metagenomics that should have detected most species regardless of their requirements.In fact, the bacterial load detected by 16S qPCR confirmed our culture findings.Second, our study assessed the effect of a PVP-I-based antiseptic solution exclusively on the anterior nares.This should be taken into consideration as S. aureus has also been found in higher and more moist areas 11,12 , where it can interact with other pathogenic microorganisms (e.g., Pseudomonas aeruginosa in chronic rhinosinusitis 37 ).The reason for limiting the application to anterior nares was to simulate the clinical situation when decolonization by any agents is limited to this area.Also, our study population was relatively young and the microbiome profile of our volunteers may be different to the older patients with comorbidities that require hospitalization 37 .Our study population did not report a history of viral infection within 14 days prior to the start of the trial.However, disruption of the nose microbiome may still be likely if a viral infection happened even before 4 weeks 38,39 , time at which the host´s immune response is also thought to recover to its baseline parameters 38 .In this regard, our results should be taken with caution as our understanding of the nose microbiome interactions with the host and external agents 40 , evolve.Moreover, our sample size was mainly determined by resource constraints rather than a formal sample size estimation.There is a possibility that our study is not adequately powered to study all the outcomes described.For example, a post hoc estimation demonstrated that our study´s power was superior to 80% for the total CFUs reduction at 5 min and 2 h, but fell to 33.6% for the data at 24 h.For this latter timepoint, the bacterial load data by 16S rRNA qPCR may be more reliable.Finally, we only tested the effect of a single intranasal application of the PVP-I antiseptic and results may vary with its repetitive use and/or additional preventive interventions.

Conclusions
Based on the findings of the current study, it appears that a single application of a PVP-I antiseptic to anterior nares is effective in reducing the bioburden of the nose and eliminating pathogens.This effect is achieved in the presence of normal mucociliary function and without disruption of the microbiome.

Study design
In order to test our hypothesis, we developed an antiseptic formulation containing povidone-iodine (PVP-I) at 10% (w/w) as the active ingredient.Then, we conducted a randomized, placebo-controlled, and parallel-group study in March 2022 (Fig. 1).
This was an unblinded study due to the discrepancy in coloration of the tested formulations with PVP-I (reddish-brown) and the phosphate-buffered saline (PBS; transparent).Nevertheless, the statistical analysis was performed by an independent, blinded statistician.The volunteers were randomized to receive PVP-I (treatment) or PBS (control) in a 1:1 ratio by block randomization.EC generated the random allocation sequence and enrolled the volunteers in this study.DFR & JC assigned and verified the interventions in our trial according to the allocation group.Sequentially numbered badges were used to identify volunteers.DFR & JC matched the badges with the allocation group before and after every intervention.The allocation group was shared to DFR & JC 5 min before the beginning of the trial.All volunteers were followed-up for up to 24 h after the application of the nasal formulation.
All methods were carried out in accordance with Title 21 of the Code of Federal Regulations (CFR), Parts 50 and 56, the Declaration of Helsinki, and Health Insurance Portability and Accountability Act (HIPAA) regulations.Western Institutional Review Board reviewed and approved this clinical study.Verbal and written informed consent were obtained from all subjects participating in this trial.Our study was also registered on the ClinicalTrials.govplatform (NCT05617729, 15/11/2022).This study was reported according to the Consolidated Standards of Reporting Trials (CONSORT) 2010 guidelines.

Participants
This population-based study comprising healthy volunteers 18 years and older was conducted at Philadelphia, Pennsylvania, USA.A total of 50 volunteer subjects were enrolled in the study.Secondary to a lack of studies assessing the longitudinal changes in microbial diversity after PVP-I application at the planification and conducting stage in our study, a formal sample size calculation was not performed.Instead, considering internal budget and the desired number of timepoints, this study was designed to include a total of 50 volunteers ( All volunteers signed a consent form and agreed to refrain from blowing and scrubbing the test area with soap, powders, lotions and/or personal care (either cosmetic or toiletry) products for 24 h after the start of this study.All follow-up visits were completed in all volunteers.

Interventions
All the interventions described in this study were performed using the same batch of antiseptic formulation containing PVP-I (#021,622-01).The clinical comparison was conducted using a negative control solution, namely PBS.
The volunteers were enrolled to calculate the bioburden at baseline, 5 min, 2 h, and 24 h post-application.Meanwhile, microbial diversity and MCC were assessed at baseline and 24 h post-application.Figure 1, summarizes all the interventions throughout the study.In addition, we collected demographic data and examined the volunteers for non-desired effects at every follow-up visit.
The swabbing and application area covered the anterior nares, the first 2-3 cm inside the nose.It consisted of a rotatory movement of the cotton tip of the swab throughout the nasal mucosa for ~ 15 s per nostril.Baseline and post-application samples were collected using Flocked FLOQSwabs (COPAN Diagnostics Inc, CA, US).After the baseline measurement, the testing solutions were applied with a standard cotton swab with polystyrene handle (Puritan, ME, USA).According to allocation, an approximate volume of 0.25 mL of the testing solution was applied per nostril.Sterile gloves were used at all times and changed between timepoints, after applying the test product (PVP-I or PBS), and between volunteers.
The flocked swabs were sealed and immediately processed to determine bioburden.With an aseptic technique, swabs were immersed in 1 mL of sterile PBS and streaked onto Trypticase Soy Agar plates.Then, swabs were stored at 4 °C until the study completion.All plates were incubated at 37 °C under aerobic conditions for 48 h.Growth was assessed by counting the colony-forming units (CFUs) at 48 h of incubation.
Likewise, nasal swabs (FLOQSwabs; COPAN Diagnostics Inc, CA, US) were sealed and sent overnight to perform NGS analysis at the MicrogenDx facility (Lubbock, Texas), similar to previous studies 41,42 .Samples were mechanically lysed using the Qiagen TissueLyser (Qiagen, Hilden, Germany).Then, each sample was spiked with a positive internal control to ensure the success of the extraction process.In addition, negative controls were run along with samples to identify contamination associated with the extraction process.DNA was then extracted using a KingFisher Flex Purification System (ThermoFisher Scientific, Waltham, MA, US).DNA was then amplified using the 28F (GAG TTT GATCNTGG CTC AG) and 388R (GCT GCC TCC CGT AGG AGT ) primers specific to the hypervariable regions V1-V2 in the 16S rRNA gene for bacteria.The Internal transcribed spacer (ITS) gene for fungi was assessed for ITS3-4 using ITS3F (GCA TCG ATG AAG AAC GCA GC) and ITS4R (GCA TCG ATG AAG AAC GCA GC) primers.The amplicons were created on a LightCycler 480 II (Roche Life Sciences, Indianapolis, IN, US) with the following thermal cycling profile: 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 52 °C for 40 s, and 72 °C for 1 min; and final extension at 72 °C for 10 min.Amplified DNA was then pooled and run on the Illumina MiSeq (Illumina Inc, San Diego, CA) at 2 × 250 bp.
We also examined MCC using the saccharin test 43,44 .A cotton swab with a wooden handle (Puritan, ME, USA) was immersed in saccharin powder (Spectrum, NJ, USA) and carefully introduced below the anterior end of the inferior turbinate.We recorded the time (minutes) between the placement of the saccharin and the volunteer´s perception of sweetness.Baseline and/or 24-h times above 30 min were considered abnormal.

Main outcomes and measures
The primary outcomes assessed were bioburden, microbial diversity and MCC (Fig. 1).Total CFU counts were determined at baseline, 5 min, 2 h, and 24 h post-application.No distinction was made between colony morphologies.CFUs were converted to log 10 and bioburden change was calculated from the baseline measure.
On the other hand, microbial diversity and MCC were assessed at baseline and 24 h post-application.Alphadiversity, which refers to the number of species or species richness detected, and beta-diversity, which refers to the number of unique species, parameters were calculated and compared between groups and timepoints.For MCC, the time to sweetness detection was reported in minutes.
In addition, as secondary outcomes, we assessed the bacterial species' relative and absolute frequency at baseline, in order to determine microbial associations, and volunteers were examined for any non-desired effects at every follow-up visit (5 min, 2 h, and 24 h post-application).

General statistical analysis
Categorical variables were summarized as absolute and relative frequencies.Comparisons were performed using the X 2 test.Quantitative data was assessed for normality with the Shapiro-Wilk test.Mean ± standard error of the mean or median (interquartile range [IQR]) were reported, accordingly.Total CFU counts were independently determined by 2 experienced members of the laboratory personnel with an inter and intra-reviewer Cohen´s kappa statistic score > 0.80.Volunteers with 1 × 10 3 or more CFUs at baseline were included in the bioburden change calculation.Comparisons were performed using the Student´s t-test.A two-tailed p-value < 0.05 was considered statistically significant.

Next-generation sequencing analysis
All analyses were conducted in R (R Core Team, http:// www.r-proje ct.org, Vienna, Austria), using the vegan 45 and phyloseq 46 packages.The operational taxonomic units (OTUs) were determined using the UPARSE

Figure 1 .
Figure 1.Study design.Fifty volunteers were recruited for this randomized clinical trial.Block randomization was performed to determine group allocation, thus, PVP-I (n = 25) or PBS (n = 25) application in the anterior nares.We assessed bioburden at baseline, 5 min, 2 h, and 24 h post-application, meanwhile microbial diversity and mucociliary clearance was assessed at baseline and 24 h post-application.Bacterial associations were assessed at baseline and non-desired events were examined at every follow-up visit.Abbreviations: PBS; phosphate-buffered saline, PVP-I; povidone-iodine.

Figure 2 .
Figure 2. Bioburden reduction (a) and mucociliary clearance assessment (b), at different timepoints after application of the testing solutions (PVP-I or PBS).(a) Data is expressed as mean ± SEM CFU log 10 reduction, from baseline bioburden, and compared using the Student t test.(b) Sweetness detection, in minutes, at baseline and 24 h post-application.This plot was performed using R (version 3.0.1,http:// www.r-proje ct.org), by the RTL Genomics facility (Lubbock, Texas).Abbreviations: PVP-I; povidone-iodine; *p < 0.05.

Figure 3 .
Figure 3. Relative abundance of the most prevalent bacterial species (a) and heatmap (b) illustrating bacteria detected to be differentially abundant between timepoints by ANCOMBC procedure.A cell colored in black can be considered "true" zero, whereas there were no detections of a particular bacteria for that given cell.This plot was performed using R (version 3.0.1,http:// www.r-proje ct.org), by the RTL Genomics facility (Lubbock, Texas).Abbreviations: PVP-I; povidone-iodine.

Figure 4 .
Figure 4. Network plot shows associations among bacterial species with p < 0.10.Each node represents a species and is linked by species associations, calculated with Spearman's rank correlation coefficient.This plot was performed using R (version 3.0.1,http:// www.r-proje ct.org), by the RTL Genomics facility (Lubbock, Texas).